Fig. 1: Proteasomal degradation controls protein levels of cancer-associated A3s.

A HEK-293T cells were transfected with different amounts of 3 x HA-tagged A3 expressing plasmids to achieve similar steady-state A3 protein levels. 24 h. post transfection, cells were treated for 16 h. with EPOX, and protein levels were analyzed by WB, and B quantified (means and SD, 2-way ANOVA, ns: p ≥ 0.05, n = 5 (for A3H-I n = 3). C RKO cells stably expressing OLLAS-A3H-I/II were fixed, and their subcellular localization determined by immunofluorescence confocal microscopy; scale bar: 20 µm. D–F Lentiviral expression constructs encoding A3H-I or A3H-II were delivered to RKO cells at different integration rates to obtain comparable A3H-I and A3H-II protein levels in the presence of proteasome inhibitor. Polyclonal cell pools were treated with CHX or MG132 for the indicated times, D protein levels analyzed by WB, E relative A3H-I and A3H-II protein levels quantified by densitometry (n = 2 biological replicates), and F single-step exponential decay curves were calculated, from which protein half-life was derived (means and SD, two-way ANOVA, corrected for multiple comparisons using the Šídák method, n = 2). G, H HEK-293T cells were transfected with equal amounts of plasmids encoding the indicated MYC-mCherry-P2A-3xHA-tagged A3H-I mutants, in which multiple lysine residues were mutated to arginine. 36 h. Post-transfection, cells were treated with EPOX for 5 h. G protein levels determined by WB, and H quantified (means and SD, multiple unpaired t-tests (two-sided), not corrected for multiple hypothesis testing, ns: p ≥ 0.05, n = 3). Source data are provided as a Source data file.